WO2018171619A1 - Procédé de détection d'activité de thiorédoxine réductase, dispositif de détection et son procédé de fonctionnement - Google Patents

Procédé de détection d'activité de thiorédoxine réductase, dispositif de détection et son procédé de fonctionnement Download PDF

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WO2018171619A1
WO2018171619A1 PCT/CN2018/079787 CN2018079787W WO2018171619A1 WO 2018171619 A1 WO2018171619 A1 WO 2018171619A1 CN 2018079787 W CN2018079787 W CN 2018079787W WO 2018171619 A1 WO2018171619 A1 WO 2018171619A1
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Prior art keywords
reagent
target
reaction
sampling
reaction cup
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PCT/CN2018/079787
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English (en)
Chinese (zh)
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尹汉维
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南京凯熙医学科技有限公司
武汉尚宜康健科技有限公司
凯熙医药（武汉）股份有限公司
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Priority claimed from CN201710172659.9A external-priority patent/CN108627469B/zh
Priority claimed from CN201710170517.9A external-priority patent/CN108627659A/zh
Application filed by 南京凯熙医学科技有限公司, 武汉尚宜康健科技有限公司, 凯熙医药（武汉）股份有限公司 filed Critical 南京凯熙医学科技有限公司
Priority to US16/496,207 priority Critical patent/US20200190556A1/en
Publication of WO2018171619A1 publication Critical patent/WO2018171619A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/02—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor using a plurality of sample containers moved by a conveyor system past one or more treatment or analysis stations
    • G01N35/04—Details of the conveyor system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/00584—Control arrangements for automatic analysers
    • G01N35/00722—Communications; Identification
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
    • G01N35/1004—Cleaning sample transfer devices
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
    • G01N35/1009—Characterised by arrangements for controlling the aspiration or dispense of liquids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N2035/00465—Separating and mixing arrangements
    • G01N2035/00514—Stationary mixing elements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/02—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor using a plurality of sample containers moved by a conveyor system past one or more treatment or analysis stations
    • G01N35/04—Details of the conveyor system
    • G01N2035/0401—Sample carriers, cuvettes or reaction vessels
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/02—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor using a plurality of sample containers moved by a conveyor system past one or more treatment or analysis stations
    • G01N35/04—Details of the conveyor system
    • G01N2035/0401—Sample carriers, cuvettes or reaction vessels
    • G01N2035/0437—Cleaning cuvettes or reaction vessels
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/02—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor using a plurality of sample containers moved by a conveyor system past one or more treatment or analysis stations
    • G01N35/04—Details of the conveyor system
    • G01N2035/0439—Rotary sample carriers, i.e. carousels
    • G01N2035/0441—Rotary sample carriers, i.e. carousels for samples
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/02—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor using a plurality of sample containers moved by a conveyor system past one or more treatment or analysis stations
    • G01N35/04—Details of the conveyor system
    • G01N2035/0439—Rotary sample carriers, i.e. carousels
    • G01N2035/0446—Combinations of the above
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/902—Oxidoreductases (1.)
    • G01N2333/90212—Oxidoreductases (1.) acting on a sulfur group of donors (1.8)

Definitions

  • the invention belongs to the field of enzyme activity detection, and particularly relates to a biochemical detection method, a detection device and an operation method thereof for the activity of thioredoxin reductase (TR) in human blood.
  • TR thioredoxin reductase
  • TR thioredoxin reductase
  • TR detection equipment can be applied to early screening of tumors in the physical examination population, tumor efficacy monitoring, recurrence warning and health management in hospitalized population, which has great market demand and development potential.
  • Patent ZL201080049877.X Method and kit for determining the activity of thioredoxin reductase in a sample and application
  • manual step-by-step operation such as: manual operation of a microplate reader, manual sample loading, Hand-operated, protected from light, manually placed on the shaker to achieve shake, manual sample loading, etc.; the various conditions of the sample are also designed for manual operation process and dosage.
  • Manual manual operation includes large manual error; various operations are non-coherent, and intermediate errors are easy to occur; long operation time is not conducive to various problems such as large-scale clinical detection applications.
  • TR detection kit in the prior art is a preliminary research and development product, and its detection steps, actions, fluxes and the like have certain limitations, so there is still room for improvement and correction in terms of detection speed and detection accuracy.
  • TR thioredoxin reductase
  • TR thioredoxin reductase
  • the object of the present invention is to provide a new fully automated thioredoxin reductase activity detecting method, detecting device and operating method thereof, thereby saving detection time and detecting steps.
  • the invention solves the problem of manually detecting thioredoxin reductase in human blood in the prior art, and can improve the detection efficiency and cost saving of thioredoxin reductase detection in clinical.
  • the invention realizes the full automatic operation of the "TR activity detection" on the biochemical detection device for the first time; the data directly obtained by the invention meets the requirements of the national detection standard, and can ensure that the detection data result reflects the early warning function of the detection technology (conformity Distribution of national data indicators).
  • a method for detecting thioredoxin reductase activity comprising: dosing, disposing a working solution, an inhibitor solution, and a mixed reagent; adding a sample, adding 50 uL to 70 uL of the working solution to the control reaction cup Add 50uL-70uL inhibitor solution to the reaction cup of the experimental group; add 10uL-30uL sample to the reaction cup of the control group and the reaction cup of the experimental group respectively; the amount of the sample is the volume required for the optimal work of the automated collaborative testing equipment. design.
  • the sample is incubated, and the control reaction cup and the experimental reaction cuvette are placed at a temperature of 30 ° C to 40 ° C for the first predetermined time in a dark environment.
  • the step of disposing the working fluid comprises: taking hydroxymethylaminomethane hydrochloride, morpholinyl propanesulfonic acid, disodium hydrogen phosphate citrate buffer system and disodium hydrogen phosphate according to a ratio of 1:1:2:4. Potassium dihydrogen phosphate buffer solution; mixing of automated synergistic detection equipment: trishydroxymethylaminomethane hydrochloride, morpholinyl propanesulfonic acid, disodium hydrogen phosphate citrate buffer system and disodium hydrogen phosphate disodium hydrogen phosphate buffer solution Evenly.
  • the automated synergistic detection device tris (hydroxymethyl) aminomethane hydrochloride has a pH of 5.5-7.2 and a concentration of 0.025-0.125 mol/L; the automated synergistic detection device has a concentration of morpholylpropanesulfonic acid of 0.25 mol/L.
  • the automated synergistic detection equipment pH of dihydrogen phosphate citrate buffer system is 2.2-8.0, the concentration is 0.2mol / L; the automated synergistic detection equipment pH of the potassium phosphate dihydrogen phosphate buffer solution is 4.9-8.2, The concentration is 1-15 mol/L.
  • the step of configuring the inhibitor solution by the automated collaborative detection device comprises: mixing the automated synergistic detection device working fluid and the inhibitor in a ratio of 1:1 to 1:5 to form an automated synergistic detection device inhibitor solution;
  • the synergistic detection device inhibitor solution is uniformly mixed, and the automated synergistic detection device inhibitor is a thioredoxin reductase inhibitor compound.
  • the step of configuring the mixing reagent by the automated collaborative testing device comprises: mixing the automated collaborative testing device reagent A and the automated collaborative testing device reagent B in a ratio of 1:4-1:8 to form an automated synergistic detecting device mixing reagent; Mix the automated synergistic detection equipment mixing reagents uniformly; the automated synergistic detection equipment A reagent is 5,5-dithiobis(2-nitrobenzoic acid) or substituted 6,6'-dinitro-3,3' - Dithiobenzoic acid; automated synergistic detection device B reagent is nicotinamide adenine dinucleotide phosphate.
  • the automated collaborative detection device has a predetermined temperature of 30-40 °C.
  • the automated collaborative detection device has a first predetermined time of 8-20 minutes.
  • the automated collaborative detection device has a first predetermined time of 10 minutes.
  • the automated collaborative detection device has a predetermined wavelength of 405 nm to 450 nm.
  • the automated collaborative detection device has a second predetermined time of 20-30 cycles.
  • the invention provides a method for detecting human peripheral blood thioredoxin reductase activity, which automatically samples and mixes reagent A and reagent B into a mixed reagent on an automated collaborative detection device, and automatically joins the sample through a synergistic detecting device.
  • the mixing and stirring operation replaces the work of separately adding the reagent A and the reagent B for manual repeated mixing and stirring, thereby improving the working efficiency.
  • the detection method can meet the requirements of the method for selecting the working fluid selection by setting the sample/reagent volume of the synergistic detecting device suitable for the thioredoxin reductase activity described above, and the method of the present invention is described above.
  • the method When used in a synergistic detecting device for thioredoxin reductase activity, the method includes a sample loading operation method, a light shielding operation request method, and an operation request method for reagent mixing, and the like, and an intelligent instruction method for a drive system.
  • the intelligent instructions such as the number of cycles in each cycle group and the time of each cycle in the operation of the collaborative detection device, and the requirements for the operation process are all detection methods that are connected with the human peripheral blood TR function detection method.
  • the method of the present invention is a method for realizing the requirement of TR enzymatic detection function by using a synergistic detecting device for thioredoxin reductase activity described above, and is used for the synergistic detection of a thioredoxin reductase activity described above.
  • a biochemical detection device for thioredoxin reductase detection comprising:
  • the accommodating device is configured to hold a plurality of reagents and/or samples, and under the driving action of the driving device, periodically rotate around the axis to rotate the target reagent and/or the target sample to the target filling hole;
  • reaction device for holding a plurality of cuvettes, and under the driving action of the driving device, periodically rotating around the axis to rotate the target cuvette to the target sampling hole;
  • a sampling device configured to periodically rotate around the axis under the driving of the driving device, and add the target reagent and/or the target sample collected from the target sampling hole to the target reaction cup corresponding to the target filling hole ;
  • a state sensing device for detecting reaction rotation state information of the reaction device, accommodation rotation state information of the accommodation device, and reagent state information of the sampling device;
  • the main control system is respectively connected to the state sensing device and the driving device, and is configured to generate and send a corresponding control instruction to the driving device based on the reaction rotation state information, the accommodation rotation state information, and the reagent state information;
  • the driving device is respectively connected to the sampling device, the accommodating device and the reaction device, and is configured to control the sampling device, the reaction device and the accommodating device to perform corresponding operations based on the received control instruction.
  • a method of operating a biochemical detection device comprising:
  • the state sensing device detects that the target reaction cup in the reaction device rotates to the target liquid adding hole and the reagent state information of the sampling device is the reagent collecting completion state, generating a reagent adding instruction
  • the driving device controls the sampling device to rotate to the target liquid adding hole of the reaction device based on the received reagent adding instruction, and adds the target reagent and/or the target sample to the target reaction cup corresponding to the target liquid adding hole;
  • the driving device controls the sampling device to rotate to the target sampling hole of the loading device to collect the target reagent and/or the target sample based on the received reagent collection instruction.
  • the present invention proposes a fully automatic biochemical detection device for detecting thioredoxin reductase (TR) in human blood, which realizes full automation of biochemical detection equipment, improves detection efficiency, and saves cost. .
  • FIG. 1 is a schematic flow chart of a method for detecting thioredoxin reductase activity of the present invention
  • FIG. 2 is a schematic structural view of a corresponding detection device for detecting a thioredoxin reductase activity of the present invention
  • FIG. 3 is a schematic flow chart of a method for detecting thioredoxin reductase activity according to a specific embodiment of the present invention
  • FIG. 4 is a schematic flow chart showing another method for detecting thioredoxin reductase activity according to a specific embodiment of the present invention
  • FIG. 5 is a flow chart of step S1 in the flow chart shown in Figure 4;
  • Figure 6 is a flow chart of step S2 in the flow chart shown in Figure 4;
  • FIG 7 is a flow chart of step S3 in the flow chart shown in Figure 4;
  • Figure 8 is a schematic diagram showing the system architecture of the biochemical detection device for thioredoxin reductase (TR) detection of the present invention
  • Figure 9 is a schematic view showing the mechanical structure of a biochemical detecting device for thioredoxin reductase (TR) detection of the present invention.
  • FIG. 10 is a schematic flow chart showing the operation method of the biochemical detecting device for detecting thioredoxin reductase (TR) according to the tenth embodiment of the present invention
  • Figure 11 is a flow chart showing the operation method of the biochemical detecting device for detecting thioredoxin reductase (TR) according to the eleventh embodiment of the present invention
  • Figure 12 is a flow chart showing the operation method of the biochemical detection device for thioredoxin reductase (TR) detection according to the twelfth embodiment of the present invention
  • Figure 13 is a flow chart showing the operation method of the biochemical detection device for thioredoxin reductase (TR) detection according to the thirteenth embodiment of the present invention
  • FIG. 14 is a schematic diagram of the detection principle of a detecting device according to an embodiment of the present invention.
  • the detection method used in the "TR activity detection kit" in the prior art (hereinafter referred to as the initial generation detection method) has three technical limitations, and thus cannot be applied to an automated collaborative detection device:
  • the initial detection method includes 7 steps and actions, which is not difficult for the manual detection of the test personnel, but is too cumbersome for the automated collaborative detection device, so the operation time of the device is significantly increased, and the detection efficiency is lowered;
  • the initial detection method requires two inspectors to cooperate with each other during the operation, and the automated collaborative detection equipment is designed to allow a single inspector to operate the instrument completely, so the requirements of the two do not match.
  • the reagent A in the present invention is an agent for detecting the activity of thioredoxin reductase
  • the reagent B is an agent for detecting the activity of thioredoxin reductase.
  • the working fluid is usually a buffer, and is mainly used as a buffer for detecting thioredoxin reductase activity.
  • the reagent A and the reagent B in the invention have all passed the expert certification and examination by the State Food and Drug Administration, and obtained the medical device registration certificate (the certificate number is the food and medicine supervision (standard) word 2013 No. 2401815 and the country Reagent A and reagent B in the thioredoxin reductase (TR) activity detection kit of Food and Drug Administration (Jun) 2014 3400264), the working fluid in the present invention is the above-mentioned "thioredoxin reduction"
  • the reagent D in the enzyme (TR) activity detecting kit, and the inhibitor in the present invention is the reagent C in the above-mentioned "thioredoxin reductase (TR) activity detecting kit".
  • Thioredoxin reductase is a reduced coenzyme II (NADPH)-dependent dimeric selenoenzyme containing a flavin adenine dinucleotide (FAD) domain, which is reduced with thioredoxin.
  • the sex coenzyme II together constitutes a thioredoxin system.
  • Thioredoxin reductase is overexpressed in cells with abnormally active proliferation, and this enzyme has physiological functions such as priming cell abnormal proliferation, initiation of apoptosis inhibition system and the like to form a tumor.
  • TR activity is highly correlated with the degree of abnormal hyperplasia of the tumor. Therefore, the detection of thioredoxin reductase has an important effect on tumors.
  • Fig. 1 is a schematic flow chart showing the method for detecting thioredoxin reductase activity of the present invention.
  • the method for detecting thioredoxin reductase activity comprises:
  • Step S1 dosing, disposing a working solution, an inhibitor solution, and a mixing reagent.
  • the working fluid is usually a buffer, and the working fluid is not particularly limited in its concentration, and is preferably formulated to have a 1X working fluid concentration.
  • the working fluid configuration process is: according to the ratio of 1:1:2:4, the reagent TrisHCL (0.025-0.125 mol / L, pH 5.5-7.2), morpholinyl propanesulfonic acid (0.25mol/L), hydrogen phosphate disodium citrate buffer system 0.2mol/L, and hydrogen phosphate disodium phosphate potassium dihydrogen phosphate buffer solution 1-15mol/L; wherein, the pH of the disodium hydrogen phosphate citrate buffer system is 2.2-8.0; pH of diammonium phosphate dipotassium phosphate buffer solution is 4.9-8.2; then buffer TrisHCL (trishydroxymethylaminomethane hydrochloride), morpholinyl propanesulfonic acid, disodium hydrogen phosphate citrate The system was uniformly mixed with a potassium hydrogen phosphate disodium hydrogen phosphate buffer solution.
  • the working solution and the inhibitor are mixed in a ratio of 1:1 to 1:5 to form an inhibitor solution; the inhibitor solution is uniformly mixed; wherein the inhibitor is a thioredoxin reductase inhibitor compound. It can be a chemical monomer selenium compound.
  • the ratio of the working fluid and the inhibitor is preferably a ratio of 1:3, and the working fluid and the inhibitor mixed at the ratio are the most economical ratio combination, that is, the subsequent human peripheral blood thioresin reductase
  • the activity detection is more accurate and is the most economical combination of reagents.
  • the reagent A and the reagent B are mixed in a ratio of 1:2-1:8 to form a mixed reagent; specifically, the mixing ratio of the reagent A and the reagent B is in the range of 1:2-1:8, preferably 1:4-5.
  • the ratio of the reagent A and the reagent B are mixed, and the mixed reagent formed by the mixing at this time has higher accuracy in the detection, wherein the A reagent is 5,5-dithiobis(2-nitrobenzoic acid) or substituted 6 , 6'-dinitro-3,3'-dithiobenzoic acid; B reagent is nicotinamide adenine dinucleotide phosphate.
  • the mixed working solution evenly; mix the inhibitor solution evenly; mix the mixing reagent evenly.
  • the mixed working solution, the inhibitor solution, and the mixed reagent are separately placed, and usually placed in a reagent tank to facilitate subsequent detection.
  • Step S2 adding a sample, adding 50 uL-70 uL of the working solution to the control reaction cup; adding 50 uL-70 uL of the inhibitor solution to the reaction cup of the experimental group; adding 10 uL to 30 uL of the sample to the reaction cup of the control group and the reaction cup of the experimental group;
  • the reaction cups of the control group and the experimental group were set at intervals, for example, the odd numbered reaction cups were the control reaction cups, and the even numbered reaction cups were the experimental group reaction cups.
  • the amount of the working fluid added to the reaction cup of the control group and the reaction cup of the experimental group is equivalent.
  • 50 uL-70 uL of the working solution is generally added to the reaction cup of the control group and the reaction cup of the experimental group.
  • 50-60 uL of working fluid is added to the control cup and the experimental set of reaction cups.
  • an equal amount of working fluid is added to the reaction cup of the control group and the reaction cup of the experimental group, so that the test data of the control group and the experimental group are comparable, and the data after the detection can be calculated.
  • the sample loading time of each sample is about 10 minutes, that is, 27 cycles, 22.5 seconds per cycle. After the sample is added, the sample of this group can be incubated, and then the other sample can be further loaded.
  • Step S3 incubating, in a dark environment, the control reaction cup and the experimental reaction cup are placed at a temperature of 30 ° C - 40 ° C for a first predetermined time; wherein, the predetermined temperature is 30 ° C - 40 ° C; The predetermined time is 8-20 minutes, preferably 10 minutes.
  • control reaction cup and the experimental reaction cup are placed at a temperature of 30 ° C to 40 ° C for 8-20 minutes in the dark, preferably 10 minutes, because the detection method can be combined with the detection.
  • the device can be used for automated detection on a synergistic detection device for thioredoxin reductase activity at a predetermined time of 10 minutes. This group of samples can be assayed after a set of samples has been incubated.
  • step S4 it is determined that 100 uL-150 uL of the mixed reagent is added to the reaction cup of the control group and the reaction cup of the experimental group, respectively; and the absorbance value in the second predetermined time period is measured at a predetermined wavelength.
  • the absorbance value in the second predetermined time period is measured at a predetermined wavelength.
  • 120uL can be added in the reaction cup of the control group and the reaction cup of the experimental group.
  • the mixed reagent continuously measures the absorbance value of the sample at a wavelength of 405 nm to 450 nm for 7.5-11.25 minutes, that is, 20-30 cycles.
  • the mixed reagent is directly added to the reagent to be measured, thereby achieving mixed mixing, which is higher than that in the prior art.
  • Reagent A and reagent B were separately stirred, and then separately applied, a part of the steps were omitted, the detection time was saved, and automatic detection on the synergistic detection device of thioredoxin reductase activity was realized, and the detection was improved. effectiveness.
  • sample in the present invention refers to any tissue derived from a living organism or a portion separated therefrom, and the sample is preferably blood, body fluid, tissue homogenate, and most preferably blood, wherein the blood may be a component such as serum, plasma or the like.
  • the detection method of the invention can be applied to "a thioredoxin activity detecting device", as shown in FIG. 2, the structure of the device is specifically:
  • Testing equipment includes:
  • the apparatus 2 includes a sample reagent disk 2-1 and a sample tube fixing member 2-2, a working fluid/inhibitor solution fixing member 2-3, and a mixed reagent fixing member 2 which are uniformly distributed in the circumferential direction of the sample reagent disk 2-1, respectively.
  • the sample tube fixing member 2-2 may be a ring-shaped structural member having a plurality of holes arranged around the circumference of the sample reagent disk, and placed in the sample reagent tray to place the sample tube, for example, a test tube rack, a sample tube
  • the number of holes in the fixing member is preferably 40;
  • the structure of the working fluid/inhibitor solution fixing member 2-3 and the mixed reagent fixing member 2-4 is similar to that of the sample tube fixing structure for respectively placing the working fluid/inhibitor solution bottle Or mixing the reagent bottles, the number is preferably 30 or 40, and each of the sample tube fixing members 2-2, each of the working fluid/inhibitor solution fixing members 2-3 and each of the mixing reagent fixing members 2-4 is Center of sample reagent plate Center, and sequentially distributed from inside to outside along the radius of the sample reagent disk.
  • the reaction device 3 is periodically rotated around the axis under the driving action of the driving system; the reaction device 3 includes: a reaction disk 3-1 and a plurality of cuvette fixing members 3-2 uniformly distributed along the circumferential direction of the reaction disk, and the reaction
  • the cup fixing part 3-2 may be an annular structural part having a plurality of holes arranged around the circumferential direction of the reaction disk, and placed in the reaction tray to accommodate or place the cuvette, for example, a test tube rack, and the number of the reaction cups is 81, can be divided into 9 groups.
  • a sampling device 4 for collecting reagents and/or samples from the accommodating device 2 into the reaction device 3 based on the periodic rotation of the accommodating device 2 and the reaction device 3;
  • the sampling device 4 is optionally a sampling needle;
  • the drive system is connected to the accommodating device 2, the reaction device 3 and the sampling device 4 for controlling the accommodating device 2, the reaction device 3 and the sampling device 4 to perform corresponding operations.
  • the hydraulic device 5 includes a vacuum pump 5-1, a vacuum tank 5-2, and a liquid path mechanism 5-3.
  • the liquid path mechanism includes a pipe and a valve disposed on the pipe, and the liquid path mechanism is also connected to the sampling device and the automatic cleaning device.
  • a vacuum pump 5-1 for adjusting the air pressure in the vacuum tank 5-2 so that the air pressure in the vacuum tank 5-2 reaches a preset air pressure
  • the vacuum tank 5-2 is configured to control the operation of the liquid path mechanism at a preset air pressure to perform an ascending, descending, and rotating operation on the sampling device, and to cause the liquid to enter or exit the automatic cleaning device.
  • the automatic cleaning device 6 is connected to the hydraulic device 5 for cleaning the reaction cup in the reaction device 3 based on the control of the hydraulic device 5; the automatic cleaning device 6 may be a cleaning needle capable of sucking liquid into the accommodation space inside the cleaning needle And then released into the reaction cup to clean the reaction cup, and then the washed waste liquid is sucked out from the reaction cup and discharged to a waste water tank (not shown), and the waste water pool can be disposed outside the detection device.
  • the stirring device 7 is connected to the driving system for agitating the mixed solution formed by the reagent and the sample, and uniformly stirring after completion of each loading step.
  • the agitation means may be a stirring needle.
  • the temperature control device 8 is located below the plurality of cuvette fixing members for controlling the temperature of the cuvette in the cuvette holding member to be maintained at the set experimental temperature.
  • the temperature control device 8 is a temperature control tank having a trough-like structure such that 81 cuvettes can be located inside the trough structure to keep the ambient temperature inside the reaction device at the reaction temperature and the incubation temperature during the reaction.
  • the optoelectronic device 9 is disposed on the upper surface of the casing 1 for controlling the optical path and the wavelength, providing illumination for the reaction process of the sample and the reagent, and continuously measuring the absorbance value of the sample.
  • the photovoltaic device is a photovoltaic box.
  • the syringe 10 is disposed on the upper surface of the housing 1.
  • the sample reagent tray is provided with a sample reagent disk cover to provide an experimental environment for the sample disk to be closed for TR activity detection;
  • the sample reagent disk cover is provided with a sample sampling hole, a working fluid/inhibitor solution sampling hole and a mixed reagent.
  • a sampling hole a sampling hole; a first liquid adding hole and a second liquid adding hole are arranged on the reaction disk; the sample sampling hole, the working liquid/inhibitor solution sampling hole, the mixed reagent sampling hole, the first liquid adding hole and the second liquid adding hole are both Located on the same circle centered on the sampling device, the sampling device is along the circumference of the circle in the sample sampling hole, the working fluid/inhibitor solution sampling hole, the mixed reagent sampling hole, the first dosing hole and the second dosing hole. Do periodic exercises.
  • the interval between the first liquid addition hole and the second liquid addition hole may be set to be less than the distance of the number of reaction cups of each set of reaction cups, preferably, the first liquid addition hole and the second liquid addition hole The interval between them can be set to a distance of 7 to 11 cuvettes, and more preferably, a distance set to 8 cuvettes.
  • the embodiment of the invention further includes a host computer, and the upper computer is connected with the hydraulic device and the driving system for transmitting an operation instruction to the hydraulic device and the driving system, so that the driving system controls the receiving device 2 and the reaction device 3 to perform periodic rotation when needed During sampling, the drive system receives the command from the upper computer to control the rotation of the accommodating device.
  • a sample tube fixing member, a working fluid/inhibitor solution bottle fixing member and a mixed reagent bottle fixing member are rotated to respectively correspond to
  • the sampling device will be lowered to the sample tube fixing member and the working fluid/inhibitor solution under the control of the hydraulic device.
  • the sample, the working liquid/inhibitor solution or the mixed reagent is collected from the sample tube, the working fluid/inhibitor solution bottle or the mixed reagent bottle, and then rises under the control of the hydraulic device, and then Move to the position of the first dosing hole or the second dosing hole and drop under the action of the hydraulic device , the collected sample, working fluid/inhibitor solution or mixed reagent is placed in the reaction cup at the first filling hole or the second filling hole position, and at the same time, the reaction device rotates one space to make the first filling liquid
  • the hole and the second liquid addition hole respectively correspond to the next reaction cup that needs to be added, and then continue to complete the collection of the next cycle and dosing.
  • a receiving device for accommodating the sample and the reagent, and periodically rotating around the axis under the driving action of the driving system
  • the reaction device for accommodating the reaction cup and the experimental cup, and the driving action of the reaction cup and the experimental cup in the driving system Next, periodically rotating around the axis; sampling means for collecting reagents and/or samples from the receiving device into the reaction device based on the periodic rotation of the receiving device and the reaction device; the driving system, respectively, and the receiving device, the reaction
  • the device is coupled to the sampling device for controlling the operation of the receiving device, the reaction device, and the sampling device.
  • the following methods are applicable to both the experimental group and the control group, and the detection methods include:
  • 3 and 4 are schematic flow charts showing the method for detecting thioredoxin reductase activity according to an embodiment of the present invention.
  • a method for detecting thioredoxin reductase activity comprises:
  • the driving system controls the sampling device to sequentially collect the working fluid/inhibitor solution from the sample reagent tray, and controls the sampling device to sequentially add the collected working fluid/inhibitor solution to the first group of reaction cups until the first One cycle group ends;
  • step S1 includes:
  • the driving system controls the reaction disk to rotate, so that the first reaction cup in the first group of reaction cups is located at the first liquid filling hole position;
  • the driving system controls the sampling device to rotate to the position of the sampling hole of the working fluid/inhibitor solution, and collects the working fluid/inhibitor solution from a working fluid/inhibitor solution bottle of the sample reagent tray;
  • the driving system controls the sampling device to rotate to the position of the first filling hole, and the collected working fluid/inhibitor solution is added into the reaction cup at the position of the first filling hole;
  • the driving system controls the sampling device and the reaction tray to repeat the adding step of the working fluid/inhibitor solution until each of the first reaction cups is added with the working fluid/inhibitor solution, and the first cycle group ends.
  • each of the preset angles may be set as the interval between the two cuvette fixing members, such that the setting can be such that each of the cuvettes in the first set of cuvettes is sequentially located in the first plus for each predetermined angle.
  • the drive system controls the sampling device to sequentially collect the working fluid/inhibitor solution from the sample reagent disk, and controls the sampling device to collect the working fluid/inhibition.
  • the solution is sequentially added to the second set of reaction cups;
  • the drive system controls the sampling device to sequentially collect samples from the sample reagent disk, and sequentially controls the sampling device to add the collected samples to the first set of reaction cups until the first cycle.
  • the second cycle group ends;
  • step S2 includes:
  • the driving system controls the reaction disk to rotate, so that the first reaction cup in the second group of reaction cups is located at the first liquid filling hole position;
  • the driving system controls the sampling device to rotate to the sampling hole position of the working fluid/inhibitor solution, collects the working fluid/inhibitor solution from a working fluid/inhibitor solution bottle of the sample reagent tray, and controls the sampling device to rotate to the first At a liquid filling position, the collected working fluid/inhibitor solution is added to the reaction cup at the first filling hole position;
  • the driving system controls the reaction disk to rotate, so that the first reaction cup in the first group of reaction cups is located at the second liquid filling hole position;
  • the driving system controls the sampling device to rotate to the position of the sample sampling hole, collects the sample from a sample tube of the sample reagent disk, and controls the sampling device to rotate to the position of the second liquid filling hole, and adds the collected sample to the second plus Inside the reaction cup at the location of the liquid well;
  • the reaction disk may or may not rotate. If the third cycle of the second cycle group is selected not to rotate, the rotation may be selected in the next cycle, but Considering the accuracy and convenience of the program control, the reaction disk preferably does not rotate; and if the sample reagent disk needs to be rotated during this cycle, it is also possible to select to rotate in this cycle or in the next cycle.
  • the drive system controls the sampling device to sequentially collect the working fluid/inhibitor solution from the sample reagent disk every two cycles, and controls the sampling device to collect the working fluid/inhibitor The solution is sequentially added to the third set of reaction cups;
  • the drive system controls the sampling device to sequentially collect samples from the sample reagent disk every two cycles, and controls the sampling device to sequentially add the collected samples to the second set of reaction cups;
  • the drive system controls the sampling device to sequentially collect the mixed reagent from the sample reagent disk every two cycles, and controls the sampling device to sequentially add the collected mixed reagents to the first set of reaction cups until The third cycle group ends.
  • the first set of reaction cups is filled with liquid and begins to enter the incubation time.
  • step S3 includes:
  • the driving system controls the reaction disk to rotate, so that the first reaction cup in the third group of reaction cups is located at the first liquid addition hole or the second liquid addition hole position;
  • the driving system controls the sampling device to rotate to the sampling hole position of the working fluid/inhibitor solution, collects the working fluid/inhibitor solution from a working fluid/inhibitor solution bottle of the sample reagent tray, and controls the sampling device to rotate to the first Adding the working liquid/inhibitor solution to the reaction cup at the first liquid addition hole or the second liquid addition hole position at a liquid addition hole or a second liquid addition hole position;
  • the driving system controls the reaction disk to rotate, so that the first reaction cup in the second group of reaction cups is located at the first liquid addition hole or the second liquid addition hole position;
  • the driving system controls the sampling device to rotate to the position of the sample sampling hole, collects the sample from a sample tube of the sample reagent disk, and controls the sampling device to rotate to the position of the first liquid filling hole or the second liquid filling hole, and the collecting is performed. a sample is added to the cuvette at the first or second dosing position;
  • the driving system controls the reaction disk to rotate, so that the first reaction cup in the first group of reaction cups is located at the first liquid addition hole or the second liquid addition hole position;
  • the driving system controls the sampling device to rotate to the position of the mixed reagent sampling hole, collects the mixed reagent from a mixed reagent bottle of the sample reagent tray, and controls the sampling device to rotate to the position of the second filling hole, and the collected mixed reagent Adding into the reaction cup at the first filling hole or the second filling hole position;
  • the working fluid/inhibitor solution, the sample and the mixed reagent are respectively added to each reaction cup in turn, until the whole plate detection is completed or the detection is stopped;
  • the dosing is cycled according to the three-cycle dosing step of the third cycle group until the working fluid/inhibitor solution, the sample and the mixed reagent are added to all the cuvettes or the detection is stopped.
  • step S4 specifically, starting from the first cycle of the fourth cycle group, the working fluid/inhibitor solution is sequentially collected every two cycles, and sequentially added to the fourth group of reaction cups; At the beginning of the second cycle, samples are taken sequentially, and the third set of reaction cups are sequentially added; from the third cycle of the fourth cycle group, the mixed reagents are sequentially collected every two cycles, and sequentially Add a second set of reaction cups;
  • the reaction device As long as the reaction device is in operation, the above step S3 can be repeated. It should be noted that after each working group is added with the working fluid/inhibitor solution, the sample and the mixed reagent, the reaction starts.
  • the reaction time is up to 22 cycles (22.5 s per cycle), preferably 20 cycles.
  • the hydraulic device can be sent by the upper computer to control the automatic cleaning device to remove the waste liquid after the reaction in the reaction cup, and the reaction cup is cleaned. After the cleaning is completed, the reaction cup is cleaned.
  • the operation of adding the working fluid/inhibitor solution, the sample, and the mixing reagent can be continued.
  • the mixture of the working fluid/inhibitor solution, the sample and the mixed reagent in the first set of reaction cups begins to react, at this time, in the next During the cycle, if the first set of reaction cups of the reaction disk are rotated to the dosing position, the sampling device is left in a waiting state.
  • the collaborative testing device performs a corresponding testing process
  • 81 reaction cups are divided into 9 groups, and each reaction has a total of 74 test cycles, including adding the first reagent to the 27 cycles of adding the sample, adding the sample to the joining 27 cycles of the two reagents, and 20 cycles of the reaction, each cycle takes 22.5 seconds, in the embodiment of the present invention
  • the sampling device takes the working fluid/inhibitor solution (working fluid or inhibitor solution) from the sampling hole position of the working fluid/inhibitor solution, and adds it to the No. 1 reaction cup of the reaction device, and then the reaction device rotates by 1 grid.
  • Spend 1 cycle (22.5s);
  • the working fluid/inhibitor solution is sequentially collected, and the working fluid/inhibitor solution is sequentially added to the 10-18 reaction cup;
  • the sampling device does not operate during the 12, 15, 18, 21, 24, 27, 30, 33, and 36 cycles.
  • the working fluid/inhibitor solution was collected sequentially at intervals of 3 cycles from the 37th cycle, and the reaction cups 19-27 were sequentially added; samples were sequentially collected every 3 cycles from the 38th cycle, and 10-19 were sequentially added.
  • the reaction cup was collected from the 39th cycle at intervals of 3 cycles, and the reaction cups 1-9 were sequentially added.
  • the working fluid/inhibitor solution is added to the No. 19 reaction cup; in the 38th cycle, the sample is added to the No. 10 reaction cup; in the 39th cycle, the mixed reagent is added to the No. 1 reaction cup; Add the working fluid/inhibitor solution to the No. 20 reaction cup; in the 41st cycle, add the sample to the No. 11 reaction cup; in the 42nd cycle, add the mixed reagent to the No. 2 reaction cup, and cycle accordingly, until the whole plate is completed or stopped. Detection
  • the cuvette group that has been previously reacted may enter the next round of inspection after washing.
  • the reaction disk can be set to rotate clockwise throughout the test
  • the reaction disk When the reaction disk is rotated clockwise, the reaction disk rotates clockwise in each cycle of the first cycle group, and the first reaction cup in the first group of reaction cups can be rotated clockwise or counterclockwise.
  • the reaction cup related to the liquid to be added cannot be rotated by a preset angle to each cycle to the first Or at the position of the second filling hole, it can be rotated clockwise to the position of the two filling holes, alternatively, it can be rotated counterclockwise.
  • the two disks work together.
  • the sample is added until the mixed reagent is added for 27 cycles (about 10min), in line with the medical requirements of TR activity detection; according to the above method to achieve detection of human blood samples, the background reduction of human blood samples by TR specific inhibitors is achieved, thereby ensuring that the detection method conforms to the detection results of TR enzyme activity and is related to the state Standard consistency (TR detection data for normal population is less than 4 units, and TR detection data for patients with high tumors is greater than 12 units).
  • the method for detecting thioredoxin reductase activity of the present invention can meet the requirements of automated detection by improving the primary detection method, and further has the following advantages:
  • the incubation time is significantly reduced, and the incubation time of a single sample is reduced from 30 minutes to 10 minutes, which can effectively reduce the detection time, so that every 40-50 samples
  • the detection can be completed within 1.5 hours, and the continuous detection of the synergistic detection equipment is realized, and the detection flux and speed requirements of the synergistic detection equipment for the thioredoxin reductase activity are met.
  • the detection action step is significantly reduced, from 7 action steps to 3-4 action steps, the detection step is optimized, and the instrument is reduced.
  • the action time and convenient instrument program operation so that a single tester can independently control the instrument to complete the entire test process.
  • the detection method of the present invention can be adapted to the action requirements, configuration requirements and instruction requirements of the TR-integrated detection device in conjunction with the thioredoxin reductase activity;
  • the clinical TR activity detection is performed on the synergistic detection device of thioredoxin reductase activity by the method of the invention, and is subjected to specific software (for details, please refer to another patent application: a thioredoxin reductase activity) Analytical method and system) treatment, can become the result of TR activity in clinical medicine, and meet the relevant requirements of the corresponding product "TR activity detection kit" and national medical device registration product standard: YZB / country (Q/CVH 001-2011 );
  • TR activity detection kit the certificate number is the food and medicine supervision (standard) word 2013 No. 2401815 and the national food medicine The supervision (probably) word 2014 3400264
  • TR detection results produced meet the relevant requirements of the corresponding products "TR activity detection kit” and national medical device registration product standards: YZB / country (Q / CVH 001-2011) .
  • the reagent A and the reagent B are automatically sampled and mixed into a mixed reagent by a synergistic detecting device for the thioredoxin reductase activity described above.
  • the synergistic detection device for thioredoxin reductase activity is automatically added to the sample for mixing and stirring, instead of adding reagent A and reagent B separately for manual mixing and stirring, thereby improving work efficiency.
  • Another object of the present invention is to provide a biochemical detection device for detecting the activity of thioredoxin reductase (TR) in human blood, which is used for realizing fully automatic biochemical detection, improving detection efficiency and saving cost.
  • TR thioredoxin reductase
  • Figure 8 is a schematic diagram showing the system architecture of the biochemical detection device for thioredoxin reductase (TR) detection of the present invention.
  • a first embodiment of the present invention provides a biochemical detection device, including: a receiving device 10, a reaction device 20, a sampling device 30, a state sensing device 50, a main control system 60, and a driving device 70.
  • the accommodating device 10 is configured to hold a plurality of reagents and/or samples, and under the driving action of the driving device 70, periodically rotate around the axis to rotate the target reagent and/or the target sample to the target liquid adding hole.
  • the reaction device 20 is configured to hold a plurality of cuvettes and, under the driving action of the driving device 70, periodically rotate around the axis to rotate the target cuvette to the target sampling hole.
  • the sampling device 30 is configured to periodically rotate around the axis under the driving of the driving device 70, and add the target reagent and/or the target sample collected from the target sampling hole to the target reaction corresponding to the target filling hole. In the cup.
  • the state sensing device 50 is configured to detect reaction rotation state information of the reaction device 20, accommodation rotation state information of the storage device 10, and reagent state information of the sampling device 30.
  • the main control system 60 is respectively connected to the state sensing device 50 and the driving device 70 for generating and transmitting corresponding control commands to the driving device 70 based on the reaction rotation state information, the accommodation rotation state information and the reagent state information;
  • Figure 9 is a schematic illustration of the mechanical structure of a biochemical detection device for thioredoxin reductase (TR) detection of the present invention.
  • the accommodating device 10 includes a accommodating tray 11 that is movably disposed to periodically rotate around the axis.
  • the accommodating tray 11 has a plurality of receiving fixtures 12 for holding reagent bottles and/or sample bottles.
  • At least one ring of the fixing member 12 is disposed on the accommodating disk 11 , and each of the rings has a plurality of accommodating fixing members 12 .
  • the number of accommodating fixtures 12 can be selected and set according to the specific needs of the user.
  • a plurality of accommodating fixing members 12 may be disposed uniformly along the edge of the accommodating tray 11, that is, each of the two accommodating fixing members 12 is spaced apart by a predetermined distance to form a uniform arrangement of at least one accommodating fixing member 12.
  • each receiving fixture 12 it can accommodate both the reagent bottle holder and the sample holder.
  • the reagent holder can be removed and replaced with the sample holder;
  • the sample holder can be removed and replaced with a reagent bottle holder, which realizes flexible exchange of the reagent position and the sample position, and can meet the different needs of different customers for the sample position and the reagent position.
  • the reaction apparatus 20 includes a reaction disk 21 which is movably disposed to periodically rotate around the axis.
  • a plurality of cuvette holders 22 for holding the cuvette.
  • At least one turn of the cuvette holder 22 is disposed on the reaction disk 21, and each of the rings has a plurality of cuvette holders 22.
  • the number of cuvettes 22 can be selected and set according to the specific needs of the user.
  • a plurality of cuvette holders 22 may be disposed uniformly along the edge of the reaction disk 21, that is, each of the two cuvette holders 22 is spaced apart by a predetermined distance to form a uniform arrangement of at least one cuvette holder 22.
  • the sampling device 30 includes a sampling rotating member 31, which is movably arranged for periodically rotating around the axis under the driving action of the driving device 70.
  • the sampling fixing member 32 is fixedly disposed on the sampling rotating member 31. Rotating under the belt action of the sampling rotary member 32; the sampling needle 33 has a fixed end at one end and a free end at the other end.
  • the main control system 60 generates a reagent addition command when receiving the reaction rotation state information when the target cuvette is rotated to the target liquid supply hole and the reagent state information is the reagent collection completion state; the driving device 70 receives the reagent addition instruction
  • the sampling device 30 is controlled to rotate to the target filling hole of the reaction device 20 and the target reagent and/or the target sample are added to the target reaction cup corresponding to the target filling hole.
  • the main control system 60 generates a reagent collection instruction when receiving the rotation state information when the target reagent and/or the target sample is rotated to the target sampling hole and the reagent state information is the reagent addition completion state; the driving device 70 is receiving After the reagent acquisition command, the sampling device 30 is controlled to rotate to the target sampling hole of the holding device 10 to collect the target reagent and/or the target sample.
  • the biochemical detection device for thioredoxin reductase (TR) detection provided by the third embodiment of the present invention further includes: a stirring device 40 for periodically rotating around the axis under the driving action of the driving device 70. The mixture formed in the target cuvette is stirred.
  • the agitating device 40 includes an agitating rotating member 41 fixedly disposed for periodically rotating around the axis under the driving action of the driving device 70.
  • the agitating fixing member 42 is fixedly disposed on the agitating rotating member. 31, the rotation of the agitating rotary member 42 is rotated; the agitating needle 43 has a fixed end at one end and a free end at the other end.
  • the main control system 60 generates a stirring control command when receiving the reagent state information as the reagent addition is completed;
  • the driving device 70 controls the stirring device 40 to rotate to the target liquid feeding hole to stir the mixed liquid in the target reaction cup.
  • the state sensing device 50 is further configured to detect the stirring state information of the stirring device 40; the main control system 60 And configured to generate a stirring reset command after receiving the stirring state information, and the driving device is connected to the stirring device 40 to control the stirring device 40 to reset.
  • the main control system 60 generates a reagent rotation command when receiving the reaction rotation state information when the number of times of rotating the predetermined angle is a predetermined number of times; after receiving the reagent rotation command, the accommodating device 10 controls the accommodating device 10 to rotate by a predetermined angle so that the next one The target cuvette is aligned with the target dosing hole.
  • the method further includes: a cleaning device 90, configured to input the liquid in the cleaning liquid into the target liquid adding hole The target cuvette is used to clean the target cuvette.
  • the cleaning device 90 includes: a hydraulic device 91 for controlling the liquid in the cleaning liquid to enter the target reaction cup or discharging the waste liquid in the target reaction cup to the target reaction cup; the cleaning assembly 92, and the hydraulic device 91 Connected, the target cuvette is cleaned by the hydraulic device 91.
  • the method further comprises:
  • the client 110 is connected to the main control system 60 for providing a user operation instruction input interface, and is configured to collect a cleaning instruction input by the user on the user operation instruction input interface, and send the cleaning instruction to the main control After receiving the cleaning command, the driving device 70 controls the cleaning device 90 to clean the target reaction cup corresponding to the target liquid filling hole.
  • the client 110 includes, but is not limited to, a host computer.
  • the method further comprises:
  • the temperature control device 80 under the driving action of the driving device 70, adjusts the experimental temperature of the reaction device 20 to maintain the experimental temperature within a predetermined experimental temperature range.
  • the state sensing device 50 is also used to collect experimental temperature data of the reaction device 20.
  • the main control system 60 is configured to: after receiving the experimental temperature data, parse the experimental temperature data to obtain a current experimental temperature, determine whether the current experimental temperature exceeds the predetermined experimental temperature range, and if so, generate a temperature reduction control instruction Or temperature rise control command.
  • the drive device 70 After receiving the temperature decrease control command or the temperature increase control command, the drive device 70 controls the temperature device to decrease or rise so that the current experimental temperature is maintained within a predetermined experimental temperature range.
  • the biochemical detection device for thioredoxin reductase (TR) detection provided in the seventh embodiment of the present invention further includes: a light source system 90 for providing predetermined experimental illumination to the cuvette in the reaction device 20 condition.
  • the invention can adopt the existing illumination adjustment technology, and details are not described herein again.
  • the biochemical detection device for thioredoxin reductase (TR) detection provided in the eighth embodiment of the present invention further includes: a power supply system 100, the main control system 60, the driving device 70, and the light source system 90 is connected to supply power to the main control system 60, the driving device 70, and the light source system 90.
  • the power system 100 includes a first power subsystem 101, a second power subsystem 102, a third power subsystem 103, and a fourth power subsystem 104.
  • the first power subsystem 101 is connected in series with the main control system 60 and the driving device 70 to supply power to the main control system 60 and the driving device 70; the second power subsystem 102 and the driving device 70 Connecting the power supply device 70 in series; the third power supply subsystem 103 is connected in series with the driving device 70 and the temperature control device 80 to supply power to the driving device 70 and the temperature control device 80; Subsystem 104, in series with the light source system 90, supplies power to the light source system 90.
  • the first power subsystem 101 is 5V DC
  • the second power subsystem 102 is 24V DC
  • the third source subsystem 33 and the fourth power subsystem 104 are 12V DC
  • the first power source The subsystem 101, the second power subsystem 102, the third power subsystem 103, and the fourth power subsystem 104 are connected in parallel.
  • the biochemical detection device for thioredoxin reductase (TR) detection provided in the ninth embodiment of the present invention further includes: a filter 4 electrically connected to the main control system 60 for a preset range of the input The internal alternating current is filtered, and the filtered alternating current is sent to the power supply system 100.
  • the alternating current in the preset range is 120V-250V.
  • Fig. 10 is a flow chart showing the operation method of the biochemical detecting device for detecting thioredoxin reductase (TR) according to the tenth embodiment of the present invention.
  • a method for operating a biochemical detection device for detecting thioredoxin reductase activity comprises:
  • Step S110 when the state sensing device detects that the target reagent and/or the target sample in the accommodating device is rotated to the target sampling hole and the reagent state information is the reagent addition completion state, generating a reagent collection instruction;
  • step S120 the driving device controls the sampling device to rotate to the target sampling hole of the loading device to collect the target reagent and/or the target sample based on the received reagent collection instruction.
  • Step S130 when the state sensing device detects that the target reaction cup in the reaction device rotates to the target liquid adding hole and the reagent state information of the sampling device is the reagent collecting completion state, generating a reagent adding instruction;
  • Step S140 the driving device controls the sampling device to rotate to the target liquid adding hole of the reaction device based on the received reagent adding instruction, and adds the target reagent and/or the target sample to the target reaction cup corresponding to the target liquid adding hole.
  • step S110 and step S120 are reagent addition processes.
  • step S130 and step S140 are reagent collection processes. Usually, the reagent is first collected and then the reagent is added, so step S120 is preceded by step S140.
  • the reagents are collected first, and the reagents are added after.
  • the signal that triggers the reagent acquisition is simply the signal that the target reagent and/or target sample is rotated to the target sampling well.
  • the signal that triggers the reagent collection is not only the signal that the target reagent and/or the target sample is rotated to the target sampling hole, but also the reagent addition completion state signal.
  • Fig. 11 is a flow chart showing the operation method of the biochemical detecting device for detecting thioredoxin reductase (TR) according to the eleventh embodiment of the present invention.
  • the method for operating the biochemical detection device for detecting thioredoxin reductase (TR) according to the eleventh embodiment of the present invention further includes:
  • Step S150 when the state sensing device detects that the reagent state information of the sampling device is that the reagent addition is completed, generating a stirring command;
  • step S160 the driving device controls the stirring device to rotate to the target liquid adding hole to stir the mixed liquid in the target reaction cup based on the received stirring command.
  • Figure 12 is a flow chart showing the operation method of the biochemical detection device for thioredoxin reductase (TR) detection according to the twelfth embodiment of the present invention.
  • the method for operating a biochemical detection device for detecting thioredoxin reductase (TR) according to the eleventh embodiment of the present invention further includes:
  • Step S170 when the state sensing device detects that the stirring state information of the stirring device is that the stirring is completed, generating a stirring reset command;
  • step S180 the driving device controls the stirring device to reset based on the received agitation reset command.
  • Figure 13 is a flow chart showing the operation method of the biochemical detection device for thioredoxin reductase (TR) detection according to the thirteenth embodiment of the present invention.
  • the method for operating a biochemical detection device for detecting thioredoxin reductase (TR) according to the thirteenth embodiment of the present invention further includes:
  • Step S210 when the state sensing device detects that the number of times the reaction device rotates by a predetermined angle is a predetermined number of times, generating a reagent rotation command
  • step S220 the driving device controls the accommodating device to rotate by a predetermined angle based on the received reagent rotation command, so that the next target cuvette is aligned with the target liquid adding hole.
  • the method further comprises:
  • the cleaning device controls the cleaning device to clean the target reaction cup corresponding to the target liquid filling hole based on the received cleaning instruction.
  • the number of cuvettes on the reaction disk is 81, which are divided into a control group and an experimental group.
  • the number of cuvettes in the experimental group was 40.
  • the 81 cuvettes are divided into 9 groups, each of which includes 9 cuvettes.
  • the accommodating disc has three rings to accommodate the fixing members, and the number of fixing members per lap is 40.
  • the first ring receiving fixing member is for holding the first reagent bottle
  • the second ring receiving fixing member is for holding the sample bottle
  • the third ring receiving fixing member is for holding the second reagent bottle.
  • the ultimate goal of the present invention is to add the first reagent, the sample, and the second reagent to all of the 81 cuvettes.
  • the first reagent, the second reagent and the sample involved in the examples of the present invention are all reagents related to the detection of thioredoxin reductase activity.
  • the sample is blood, body fluid or tissue homogenate.
  • the first reagent includes a working solution (a mixture solution of TrisHCL, morpholinylpropanesulfonic acid, disodium hydrogen phosphate citrate buffer system, and disodium hydrogen phosphate disodium hydrogen phosphate buffer solution) and a thioredoxin reductase inhibitor compound. .
  • the second reagent is a mixed reagent comprising 5,5-dithiobis(2-nitrobenzoic acid) or substituted 6,6'-dinitro-3,3'-dithiobenzoic acid and nicotinamide adenine A mixed solution of dinucleotide phosphoric acid.
  • the working fluid configuration process is: according to the ratio of 1:1:2:4, the reagent TrisHCL (0.025-0.125 mol / L, PH 5.5.8-7.2), morpholinyl C Sulfonic acid (0.25mol/L), disodium hydrogen phosphate citrate buffer system 0.2mol/L, and sodium hydrogen phosphate disodium phosphate potassium dihydrogen phosphate buffer solution 1-15mol/L; wherein the hydrogen phosphate disodium citrate buffer The pH of the system is 2.2-8.0; the pH of the potassium hydrogen phosphate disodium phosphate buffer solution is 4.9-8.2; then the TrisHCL (trishydroxymethylaminomethane hydrochloride), morpholinyl propanesulfonic acid The disodium hydrogen phosphate citrate buffer system and the potassium hydrogen phosphate disodium hydrogen phosphate buffer solution are uniformly mixed.
  • the working solution and the inhibitor are mixed in a ratio of 1:1 to 1:5 to form the inhibitor solution; the inhibitor solution is uniformly mixed; wherein the inhibitor is thioredoxin reductase inhibition Compound. It can be a chemical monomer selenium compound.
  • the mixed reagent generated by the mixing is highly accurate at the time of detection, wherein the A reagent is 5,5-dithiobis (2- Nitrobenzoic acid) or substituted 6,6'-dinitro-3,3'-dithiobenzoic acid; B reagent is nicotinamide adenine dinucleotide phosphate.
  • a reagent is 5,5-dithiobis (2- Nitrobenzoic acid) or substituted 6,6'-dinitro-3,3'-dithiobenzoic acid
  • B reagent is nicotinamide adenine dinucleotide phosphate.
  • the present invention provides three liquid sampling holes, and the first reagent corresponds to the first liquid sampling hole, and the sample corresponds to the first The second liquid collecting hole corresponds to the third liquid collecting hole.
  • the present application provides two liquid addition holes (the first liquid addition hole and the second liquid addition hole).
  • the first cycle group only the first reagent is added to the first reaction cup, and the first reagent may be added through the first liquid addition hole or the second liquid addition hole.
  • the second cycle group a sample is added to the first set of reaction cups, and a first reagent is added to the second set of reaction cups, and the first reagent and the sample can be added through the first liquid addition hole and the second liquid addition hole.
  • a second reagent is added to the first reaction cup, a sample is added to the second reaction cup, and a first reagent is added to the third reaction cup, and then, according to the liquid addition sequence of the third group, continue to The first set of reagents, the sample and the second reagent are added to the second set of cuvettes to the ninth set of reaction cups.
  • the driving device controls the rotation of the accommodating disk, and when the state detecting device 50 detects that the first reagent in the accommodating device rotates to the first liquid filling hole or the second liquid adding hole, and the reagent state information When the reagent is added to the completion state, a first reagent collection instruction is generated;
  • the driving device controls the sampling device to rotate to the first sampling hole of the holding device to collect the first reagent based on the received first reagent collecting instruction;
  • the driving device 70 controls the sampling device 30 to rotate to the first liquid filling hole or the second liquid adding hole of the reaction device 20 and add the first reagent to the first liquid filling hole or the first The second addition liquid hole corresponds to the first reaction cup.
  • the driving device controls the reaction device 20 to rotate, and when the state sensing device 50 detects that the first cuvette in the second group of reaction cups in the reaction device rotates to the first liquid addition hole
  • the reagent state information of the sampling device is the reagent collection completion state
  • a first reagent addition command is generated.
  • the driving device controls the sampling device to rotate to the first liquid adding hole of the reaction device based on the received reagent adding instruction, and adds the first reagent to the first reaction cup corresponding to the first liquid adding hole;
  • the driving device controls the sampling device to rotate to the first sampling hole of the receiving device to collect the first reagent based on the received first reagent collecting instruction;
  • the driving device 70 controls the sampling device 30 to rotate to the first liquid adding hole of the reaction device 20 and adds the first reagent to the first reaction cup corresponding to the first liquid filling hole.
  • the sampling device collects the sample according to the process of collecting the first reagent, adds the first reagent in the first cycle of the second cycle group, and adds the sample in the second cycle until the nine reaction cups in the second reaction cup are added.
  • the first reagent, and the nine cuvettes in the first set of cuvettes are added to the sample, and the second cycle group ends.
  • a first reagent is added to the third set of reaction cups; in the second cycle, a sample is added to the second set of reaction cups; and in the third cycle, a second reaction is added to the first set of reaction cups. Reagents until 9 cuvettes in the third set of cuvettes are added to the first reagent, 9 cuvettes in the second set of cuvettes are added to the sample reagent, and 9 cuvettes in the first set of cuvettes are added to the second reagent
  • the third cycle group ends.
  • the first reagent, the sample and the third reagent are added to the reaction cups of Groups 4-9 until the whole plate detection is completed or the detection is stopped.
  • reaction time is up to 22 cycles (22.5 s per cycle), preferably, For 20 cycles, for the set of cuvettes, if the set of reaction cups is rotated to the dosing position during the 22 cycles, the pipetting device is left in a waiting state.
  • the hydraulic device can be controlled by the client, and then the cleaning device can be controlled to remove the waste liquid in the reaction cup, and the reaction cup is cleaned. After the cleaning is completed, the reaction cup is The operation of adding the first reagent, the sample, and the second reagent can be continued.
  • the mixture comprising the first reagent, the sample and the second reagent in the first set of reaction cups begins to react, at this time, in the next cycle. If the first set of reaction cups of the reaction disk are rotated to the dosing position, the pipetting device is left in a waiting state.
  • biochemical detection device of the invention can not only detect biochemical reactions, but also detect non-biochemical reactions, it is only necessary to input biochemical reaction parameters or non-biochemical reaction parameters to be input on the user input interface of the client. Automatic adjustment according to the specific biochemical reaction or non-biochemical reaction detection process.
  • the present invention aims to provide a method for detecting thioredoxin reductase activity, which is capable of conforming to operation by setting a sample/reagent volume suitable for a synergistic detecting device for thioredoxin reductase activity described above.
  • the method for selecting a working fluid requires the method of the present invention to be used on a synergistic detecting device for thioredoxin reductase activity described above, including a method for loading a sample, a method for protecting from a light operation, and a method for operating a reagent mixture.
  • Such as the intelligent instruction method for the drive system is provided.
  • the intelligent instructions such as the number of cycles in each cycle group and the time of each cycle in the operation of the collaborative detection device, and the requirements for the operation process are all detection methods that are connected with the human peripheral blood TR function detection method.
  • the method of the present invention is a method for realizing the requirement of TR enzymatic detection function by using a synergistic detecting device for thioredoxin reductase activity described above, and is used for the synergistic detection of a thioredoxin reductase activity described above.
  • a method for detecting thioredoxin reductase activity of a device is a method for realizing the requirement of TR enzymatic detection function by using a synergistic detecting device for thioredoxin reductase activity described above, and is used for the synergistic detection of a thioredoxin reductase activity described above.
  • Another object of the present invention is also to protect a biochemical detection device for biochemical detection of thioredoxin reductase in human blood and an operation method thereof.
  • the biochemical detection device of the present invention realizes sulfur in human blood for the first time.
  • the fully automated detection of oxyredoxin reductase activity solves the problem of intelligent detection of thioredoxin reductase activity by manual in the prior art; on the other hand, the biochemical detection device of the present application can not only realize a single sample set TR The inspection process also enables multiple samples to be continuously completed in the TR detection process.
  • the completion of the overall detection process for a single sample can be achieved. Due to the continuous processing of the sample, the continuous loading and reaction time are strictly required. Since the sample disk rotates two squares, the reaction disk rotates one space, that is, two samples are taken from each sample tube, and two reaction cups are respectively added as an experimental group. And the detection of the data of the control group, so that the simultaneous detection of a single sample can be completed.
  • the hardware settings include the configuration of the reaction disk, the configuration of the sample disk, the linkage mode between the two disks, the sample loading and sampling device, etc., which can realize the completion of the multi-sample continuous detection process.
  • the improvement of the method includes the timing of the rotation time, the rotation interval, the rotation distance, the sampling sequence, the loading time and the like for the sample tray and the reaction tray. Since each reaction cup needs to add three times of solution, it can be reacted in each group. During the process of adding the solution to the cup, the addition of the next set of reaction cups is started at the same time, so that a plurality of samples can be continuously detected and can be cyclically detected, thereby shortening the detection time.
  • the present invention employs specific driving hardware, enabling software and hardware to work together, which can improve clinical TR detection efficiency and save costs.
  • the experimental results detected by the biochemical testing device of the present invention can meet the requirements of national testing standards.

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Abstract

L'invention concerne un procédé de détection d'activités d'une thiorédoxine réductase, un dispositif de détection et un procédé de fonctionnement associé. Le procédé de détection comprend : la préparation d'une solution : la préparation d'une solution de travail, d'une solution d'inhibiteur et d'un agent mixte ; l'addition d'un échantillon : l'ajout de la solution de travail dans une coupelle de réaction de commande, l'ajout de la solution d'inhibiteur dans une coupelle de réaction expérimentale, et l'ajout respectif d'un échantillon dans la coupelle de réaction de commande et la coupelle de réaction expérimentale ; une incubation : mise en place de la coupelle de réaction de commande et de la coupelle de réaction expérimentale dans un environnement sombre pour effectuer une incubation à une température prédéterminée pendant un premier temps prédéterminé ; et une mesure: l'ajout de l'agent mélangé dans la coupelle de réaction de commande et la coupelle de réaction expérimentale et la mesure de la valeur d'absorbance à une longueur d'onde prédéterminée pendant un second temps prédéterminé. Ledit procédé de détection et le dispositif de détection peuvent réaliser une détection entièrement automatisée des activités de la thiorédoxine réductase, de manière rapide et efficace, tout en faisant des économies de temps et d'effort.
PCT/CN2018/079787 2017-03-21 2018-03-21 Procédé de détection d'activité de thiorédoxine réductase, dispositif de détection et son procédé de fonctionnement WO2018171619A1 (fr)

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CN201710172659.9A CN108627469B (zh) 2017-03-21 2017-03-21 一种用于协同检测设备的硫氧还蛋白还原酶活性检测方法
CN201710170517.9A CN108627659A (zh) 2017-03-21 2017-03-21 硫氧还蛋白还原酶检测的生化检测设备及其操作方法
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